Method for the detection of cancer

ABSTRACT

A METHOD IS PROVIDED FOR DETERMINING WHETHER CANCER IS PRESENT IN A TISSUE. THIS METHOD MAKES USE OF THE BIOLUMINESCENT REACTION BETWEEN ATP AND FIREFLY LANTERN EXTRACT AND MEASURING THE AMOUNT OF LIGHT WHICH IS EMITTED. A COMPARISON IS MADE BETWEEN CELLS TAKEN FROM THE SUBJECT WHICH ARE KNOWN TO BE NONCANCEROUS AND THE CELLS WHICH ARE THOUGH TO BE CANCEROUS. THE PRESENCE OF CANCER IS INDICATED BY A SIGNIFICANTLY LESSER AMOUNT OF LIGHT BEING EMITTED BY THE SAMPLE WHICH IS SUSPECTED TO CONTAIN CANCER.

United States Patent 3 575,811 METHOD FOR THE DETECTION OF CANCER EmmettW. Chappelle, Baltimore, and Gilbert V. Levin,

Chevy Chase, Md., assignors to Hazleton Laboratories,

Incorporated, Falls Church, Va.

No Drawing. Continuation of application Ser. No. 433,461, Feb. 17, 1965.This application Oct. 23, 1968, Ser. No. 770,906

Int. 'Cl. G01n 31/14 US. Cl. 195103.5 4 Claims ABSTRACT OF THEDISCLOSURE A method is provided for determining whether cancer ispresent in a tissue. This method makes use of the bioluminescentreaction between ATP and firefly lantern extract and measuring theamount of light which is emitted. A comparison is made between cellstaken from the subject which are known to be noncancerous and the cellswhich are thought to be cancerous. The presence of cancer is indicatedby a significantly lesser amount of light being emitted by the samplewhich is suspected to contain cancer.

This is a continuation of SN. 433,461 filed Feb. 17, 1965, and nowabandoned.

This invention relates to a method for the detection of cancer. Moreparticularly, this invention relates to the use of the bioluminescentreaction, between adenosine triphosphate (hereinafter referred to asATP) and firefly lantern extract as a means for detecting the presenceof cancerous cells.

It is known that the energy requirements for all biological reactionsare directly or indirectly supplied through ATP which is present in allliving tissue cells. It has now been found that the ATP content ofcancerous cells is only /3 to or less, the ATP content of normal cells.

It is an object of this invention to provide a method for the rapiddetection of cancerous cells by monitoring the ATP content of the cells.

It is a further object of this invention to provide a means for theutilization of the bioluminescent reaction between ATP and fireflylantern extract as a means for detecting the presence of cancerouscells.

Firefly lantern extract contains a mixture comprising luciferin,luciferase and the cation, magnesium. When a material containing ATP isbrought into contact with firefly lantern extract, there is a reactionaccompanied by the emission of light. This emission of light is causedby the reaction of ATP with the constituents of the firefly lanternextract in the presence of oxygen. This phenomenon ies called fireflybioluminescence.

The reactants required for firefly bioluminescence are r Mg ATPluciferin luciieryl adenylate Pyrophosphate luciierase O2Luciferyladenylate oxylnciferyladenylate light, H2O

lucilerase This reaction is absolutely specific for ATP. The ATP may notbe replaced by any other known compound.

ice

The objects of this invention are attained, briefly, by a process whichcomprises the utilization of the bioluminescent reaction between ATP andfirefly lantern extract. A sample of the tissue suspected to containcancer is prepared, for example, by comminuting it and suspending it indistilled water. An equal amount of a sample of the adjacent tissue,such as obtained from the same animal and organ, which is known to benon-cancerous, is prepared in the same manner. An equal quantity of eachof these samples is then mixed, in the presence of oxygen, with fireflylantern extract which includes a mixture comprising luciferin,luciferase and a cation such as magnesium. A reaction will occur whichis accompanied by the emission of light. This phenomenon is fireflybioluminescence. The quantity of light is measured and the amount oflight emitted by the tissues suspected to contain cancer is comparedwith the amount of light emitted by the tissue known to be noncancerous.The presence of cancer in the unknown material will be indicated by asignificantly less amount of light being emitted by the tissue suspectedto contain cancer than the tissue known to be noncancerous on an equalaliquot basis.

The firefly bioluminescent reaction may be carried out utilizing crudefirefly lantern extracts or the purified constituents therefrom whichparticipate in the bioluminescent reaction. It has been found that asufficiently high degree of sensitivity may be attained using theprimary extract of the firefly lantern.

Lyophilized firefly lantern extract may be obtained commercially. Thismaterial may be prepared for use by dissolving it in distilled,deionized water to the desired concentrations such as, for example, bydissolving 70 mg. of lyophilized firefly lantern extract in 5 ml. ofwater. The lyophilized preparation will also contain MgSO and potassiumarsenate in amounts suflicient to result in concentrations of 0.01 M and0.05 M, respectively. The pH of such a solution is 7.4. The solutionsmay be further diluted to give any desired concentration of fireflylantern extarct.

The firefly lantern extract which mey be used in the practice of thisinvention may also be prepared in the laboratory from desiccated fireflytails. The firefly tails are first ground to a fine powder with a mortarand pestle with a small amount of washed silica. The powder is thenextracted with 0.05M potassium arsenate-0.0l M MgSO at pH 7.4.

When crude firefly lantern extract is used in the practice of thisinvention, there may be present in the extract small amounts of ATP andATP precursors, along with phosphorylating enzymes capable of convertingthe precursors into ATP. The presence of these materials may give riseto a basal light emission by the firefly extract in the absence ofexogenous ATP. This type of light emission, which is referred to asinherent light, occasionally may interfere with the detection of lightemission in the practice of this invention. However, the problem ofinherent light may be eliminated or minimized by one or more of thefollowing techniques:

(1) The firefly extract may be partially purified to remove the factorsresponsible for the inherent light. The separation and partialpurification of luciferase and luciferin is described by McElroy(Methods in Enzymology, vol. II, page 851, Academic Press, Inc., NewYork, 1955).

(2) Another approach to the removal of inherent light involves thesalting out of luciferase by the addition of ammonium sulfate to thefirefly extract, leaving the non-protein factors responsible forinherent light in the supernatant. This has been accomplished asfollows: mg. of lyophilized extract were suspended in 10 ml. of 2.7 Mammonium sulfate. After standing at room temperature for 15 minutes, thesuspension was centrifuged at approximately 200 G for minutes afterwhich the supernatant was discarded. The precipitate, after being washedtwice with 10 ml. aliquots of 2.7 M ammonium sulfate, was taken up in2.5 ml. of a solution of 0.05 M potassium arsenate buffer (pH 7.4) and0101 M magnesium sulfate. This treatment, followed by reconstitutionwith partially purified luciferin, reduces the overall activity of theextract by only about and reduces the inherent light by about 90%.

(3) Experiments have indicated that the use of calcium phosphate gelwill also reduce the inherent light in the extract Without significantlyreducing the activity of the extract. Thus, 50 mg. of commerciallyophilized firefly extract were dissolved in 1.25 ml. of deionizedwater and centrifuged. The solution was then treated from one to threetimes with varying amounts of calcium phosphate gel (from 249 mg. to 334mg.). The treatment consisted of shaking the gel with the extract forten minutes and then removing the gel by centrifugation at 200 G for 10minutes. With one treatment with calcium phosphate gel, there is areduction of about 85% of the inherent light with only a loss of from 7to 26% of luciferinluciferase activity.

(4) The simplest means for reducing the inherent light is by dilution ofthe extract with water. Maximum sensitivity with the least amount ofinherent light is obtained at a lyophilized extract concentration of 3mg./ml.

The practice of this invention may be used to determine rapidly thepresence of cancer present in small quantities of tissues. Usingelectronic equipment, tissues or cells containing an amount of ATP lessthan 2 10- ig. and approaching 10- ,ug. may be assayed. Thebioluminescent reaction itself takes less than 0.5 second to attainmaximum amplitude.

Any animal tissue may be tested for cancer by the practice of thisinvention. The process may be used as a diagnostic tool permitting therapid determination of a maligancy in clinical or surgical cases. Thisnew method is more rapid than the present biopsy procedures and hencewill appreciably decrease the lapse of time required for the diagnosisof patients undergoing surgery and, consequently, the time required forthe operation. This invention may also be used in research inconjunction with the detection of virus as described and claimed inconcurrently filed application Ser. N 0. 433,462 filed Feb. 17, 1965,and now abandoned, for Method for the Detection of Virus." The methodmay permit a determination of the viral or nonviral origin of specificcancer. The method may also facilitate a study of the kinetics ofmalignant growth with a view towards elucidating the mechanism and topermit possible control.

In assaying the samples of tissue, results may be obtained by mixing theintact tissue cells with the firefly lantern extract. However, formaximal response and in order to make more accurate assays, it ispreferred to comminute the tissue and to rupture the cells and extractthe ATP therefrom. A variety of method for the extraction of ATP fromthe cells may be used. These include hot Water extraction, acetoneextraction, ultrasonic disruption, dimethylsulfoxide extraction andperchloric acid extraction. Some of the methods which may be used foraccomplishing rupture and extraction of the cells are described below:

(A) Acetone and hot water: One ml. of the comminuted tissue is added to10 ml. of deionized water and the suspension maintained at 100 C. forone to five minutes. The preparation is then cooled and assayed for ATP.

The acetone extraction consists of adding one ml. of comminuted tissuesuspension to ten ml. of acetone. After standing for one to fiveminutes, a one ml. aliquot of the mixture is taken to dryness in air andthe residue suspended in one ml. of deionized water. The preparation isthen assayed.

(B) :Dimethyl sulfoxide (DMSO): DMSO is a highboiling point organicsolvent, miscible with water in all proportions and exhibiting a verylow order of toxicity. One ml. aliquots of comminuted tissue suspensionare added to ten ml. of various concentrations of DMSO in water. Afterstanding for five minutes, the suspension is assayed for ATP response.

(C) Ultrasonic oscillation: Ultrasonic oscillation has been successfullyused by many investigators for the rupture of cells. Five ml. of anaqueous suspension of finely divided tissue are subjected to one to fiveminutes of ultrasonic oscilaltion at 50 to Watts. After cooling the tubein flowing water, the treated suspension is assayed for ATP response.Trichloroacetic acid (0.5 ml. of 5% solution) may be added to thesuspension prior to sonification in order to stabilize the ATP againsthydrolysis.

(D) Perchloric acid: Up to 0.2 ml. of perchloric acid may be added to 5ml. of tissue suspension. The prepara tion is then assayed.

It is preferred to contact the comminuted tissue to be tested and thefirefly lantern extract in a liquid reaction medium, such as sterile,deionized water. The liquid reaction medium will generally containenough dissolved oxygen to allow the bioluminescent reaction to takeplace.

The material to be assayed for ATP content should be mixed with thefirefly lantern extract in a manner which permits the mechanicalmeasurement 'and recordation of the light emitted. The amount of lightemitted is directly proportional to the total ATP content of thematerial being tested other conditions being equal. Comparisons ofnormal and malignant tissue are made on the basis of ATP content perunit tissue volume or weight or per unit protein content, or anycombination thereof.

The procedure for using the instruments which are used to detect andrecord the intensity of emitted light consists of injecting a liquidmedium containing the material to be assayed, such as an aqueous extractof the material, into a cuvette containing the firefly latern extract.The extract is held at pH 7.4 with potassium arsenate buffer. The lightemitted as the result of the reaction between the ATP in the tissueculture to be tested and the firefly lantern extract strikes the surfaceof a photomultiplier tube giving rise to a current which can be measuredand recorded by either an oscilloscope photograph or a linear recorder.The unit of intensity used for comparing these reactions is themillivolt. The unit of light intensity has been arbitrarily defined asbeing equivalent to one millivolt. Alternately, a pulse counting devicewith a digital or analogue read-out may be used to record the reaction.

Because the response (i.e., light emission) is almost instantaneous whenthe tissue culture is contacted with the firefly lantern extract, theextract should be positioned in front of the light detection systemprior to the inroduction of the material to be assayed.

There are two ways in which the bioluminescent response with ATP presentin a material can be expressed. One is by measurement of the maximumintensity of the emitted light, which after reaching this maximum value,decays exponentially. With all other factors constant, the maximumintensity is directly proportional to the concentration of ATP. Thealternative manner of expressing the response is by integration of thetotal amount of light emitted; i.e., area under the light intensitycurve. This is the slower of the two methods, because of the relativelylong time necessary for complete decay (up to 10 minutes). Therefore,maximum intensity has been chosen as the measure of ATP response.

The instrumentation necessary for the quantitative measurement ofbioluminescence consists of a photomultiplier tube for the conversion oflight energy into an electrical signal, a device for determining themagnitude of the signal, and a light tight chamber for presentation ofthe bioluminescent reaction to the photomultiplier tube.

In one system, part of the assembly consists of a composite sensing andreaction chamber which contains a photomultiplier tube, with appropriatecircuitry, and a rotary cylinder mounted in a block of aluminum in amanner which permits removal of the reaction chamber with out exposingthe phototube to light. A section of the cylinder wall is cut out toaccommodate a standard ten mm. or five mm. rectangular cuvette.Immediately above the cuvette holder is a small injection port sealedwith a replaceable light-tight rubber plug. The entire unit is paintedblack to reduce light reflection. The photomultiplier converts the lightenergy into an electrical signal. An oscilloscope, which records themagnitude of the signal from the photomultiplier, is provided with amaximum sensitivity of 200 v./cm. of beam deflection which will allow anincrease in system sensitivity by decreasing the bandwidth or directlyreducing the noise level. There is a multiple switching arrangement atthe scope input Which makes it convenient to adjust the system zeros andbalances. The differential input to the scope provides a means tobalance the dark current output of the phototube. The response to thefirefly luminescent system displayed on the oscilloscope screen isrecorded with a camera which mounts directly onto the front of theoscilloscope. To observe and record the reaction, the cuvette containingthe necessary reagents is positioned in the cuvette carrier withoutexposing the phototube. Rotation of the carrier positions the cuvette infront of the phototube. The unknown, is then added through the injectionport and the magnitude of the response, if any, is recorded by thecamera.

The overall sensitivity and perhaps reliability of the bioluminescentreaction of the material to be tested may be increased by the conversionof other nucleotide phosphates which are present in tissues, such asadenosine diphosphate (ADP) and adenosine monophosphate (AMP), to ATP.This may be accomplished as described below by adding to the tissueculture certain phosphorylating enzymes. One such enzyme isphosphocreatine kinase.

Phosphocreatine kinase (1030 units/mg.) is prepared in a concentrationof 0.4 mg./ml. in 0.05 M potassium arsenate bufier (pH 7.4) containingMgSO at concentration of 10- M and creatine phosphate at a concentrationof 0.1 mg./ ml. One tenth ml. of this solution is added to 1 ml. of asolution being assayed for ADP. The mixture is allowed to incubate at 30C. for 30 minutes at room temperature and is then boiled for fiveminutes to destroy the kinase. After cooling at 0 C., a ml. aliquot isremoved for ATP assay. The difference between the ATP content prior toincubation with phosphocreatine kinase and the ATP content after suchincubation is the amount of ADP which has been converted to ATP.

The following examples illustrate the practice of the invention.

EXAMPLES 1 TO 4 In these examples, different tissues are obtained inboth cancerous and noncancerous form. The noncancerous tissues are takenfrom the same animals and organs as those containing the malignancy andare used as controls. The cancerous tissues contain the type of tumorousgrowth listed in Table I.

A quantity of commercially obtained lyophilized firefly lantern extractcorresponding to 50 mg. of firefly tails is dissolved in ten ml. ofdistilled water. After centrifugation at 1700 x G to remove debris, thesolution, which contains luciferase, luciferin, MgSO l0 M, and arsenatebuflfer 10 M, is ready for use. One ml. of the extract is placed into acuvette which is then positioned in the light detection chamber of anapparatus as previously described. Suflicient dissolved oxygen for thebioluminescent reaction is present in the extract.

Before the tissues containing unknown concentrations of ATP are assayed,a calibration of known concentrations of ATP is plotted by injecting Aml. portions of known concentrations of ATP through the light proof sealinto the cuvette by means of a hypodermic syringe. The light response inmillivolts is plotted against the ATP concentration. A straight linearfunction is obtained. For example, the response from 10* gamma of ATP is20,000 millivolts while that from 10- is 2000 millivolts, etc.

A sample (500 mg.) of each tissue is minced with a scalpel and suspendedin a solution composed of 4.5 ml. of distilled water and 0.5 ml. of 2%(by weight) trichloroacetic acid. The suspension is boiled for 5 minutesand then cooled to approximately 0 C. in an ice bath. Each sample isthen brought up to a volume of 6 ml. with distilled water after which itis subjected to ultrasonic rupture at approximately 75 watts power forone to five minutes. The samples are then cooled to approximately 0 C.,in an ice bath. One ml. of each sample is diluted to a final volume of10 ml. with distilled water. One tenth ml. aliquots of each sample isassayed for ATP content using the firefly bioluminescence technique byinjecting the aliquot into the cuvette of the apparatus and measuringthe amount of light emitted. The results are set forth in Table I. TheATP content, in this example, is expressed in terms of micrograms of ATPper milligram of wet tissue It is seen that the ATP content of thecancerous tissue is considerably lower than the correspondingcontrols--i.e., non-cancerous tissues.

EXAMPLES 5 TO 9 The mouse and monkey tissues listed in II which aresuspected to be cancerous are obtained. Corresponding tissues which areknown to be noncancerous are obtained from the same animal and organs asthe suspected malignant tissues. The tissues are all assayed for ATPcontent by the process described in Examples 1 to 4. In each example,the ATP content of the tissue suspected to be cancerous is significantlylower than the corresponding non-cancerous tissue, ranging from 10 to30% of that present in equal aliquots of the non-cancerous tissue. Thisconfirms the suspected presence of cancer in the cells tested.

TABLE 11 Example: Tissue 5 Erythrocytes (mouse). 6 Leucocytes (mouse). 7Axillary region (mouse). 8 Liver, kidney (mouse). 9 Kidney (monkey).

We claim:

1. A method for determining the presumptive presence of cancer in atissue which comprises preparing a sample of the tissue suspected tocontain cancer, preparing an equal quantity of a sample of adjacenttissue from the same animal and organ which is known to be noncancerous,mixing each of said tissues in the presence of oxygen with fireflylantern extract, said extract containing luciferin, luciferase andmagnesium ions, measuring the amount of light emitted by the reaction ofeach of said samples with said extract, comparing the amount of lightemitted by said sample suspected to contain cancer with the amount oflight emitted by said sample which is known to be noncancerous, thepresence of cancer being indicated by a significantly lesser amount oflight being emitted by said sample suspected to contain cancer than saidtissue known to be noncancerous.

2. A method for determining the presumptive presence of cancer in ananimal tissue which comprises obtaining a sample of the tissue suspectedto contain cancer and another sample of tissue from the same animal andorgan which is known to be noncancerous, comminuting each of saidsamples, preparing an aqueous suspension of equal amounts of each ofsaid comminuted samples, treating each of said suspensions to rupturethe cells of said tissues' and extracting the adenosine triphosphatetherefrom, thereafter mixing said treated suspensions of sample in thepresence of oxygen with firefly lantern extract, said extract containingluciferin, luciferase and magnesium ions measuring the amount of lightemitted by the reaction of each of said suspensions of sample with saidextract, comparingthe amount of light emitted by said suspension ofsample suspected to contain cancer with the amount of light emitted bysaid suspension of sample which is known to be noncancerous, thepresence of cancer being indicated by a significantly less amount oflight being emitted by said suspension of sample suspected to containcancer than said suspension of tissue known to be noncancerous.

3. The method of claim 2 Wherei said suspensions are subjected toultrasonic oscillation in order to rupture the cells of said tissues.

4. The method according to claim 2 wherein the amount of light emittedis increased by the addition of phosphocreatine kinase.

References Cited UNITED STATES PATENTS 3,099,605 7/1963 Free 195-1035ALVIN E. TANENHOLTZ, Primary Examiner US. Cl. X.R. 19566, 127

